Abstract

Voltage-gated potassium (Kv) channels regulate many physiological functions and represent important therapeutic targets in the treatment of several clinical disorders. Although some of these channels have been well-characterized, the study of others, such as Kv3 channels, has been hindered because of limited pharmacological tools. The current study was initiated to identify potent blockers of the Kv3.2 channel. Chinese hamster ovary (CHO)-K1 cells stably expressing human Kv3.2b (CHO-K1.hKv3.2b) were established and characterized. Stichodactyla helianthus peptide (ShK), isolated from S. helianthus venom and a known high-affinity blocker of Kv1.1 and Kv1.3 channels, was found to potently inhibit 86Rb+ efflux from CHO-K1.hKv3.2b (IC50 ∼ 0.6 nM). In electrophysiological recordings of Kv3.2b channels expressed in Xenopus laevis oocytes or in planar patch-clamp studies, ShK inhibited hKv3.2b channels with IC50 values of ∼0.3 and 6 nM, respectively. Despite the presence of Kv3.2 protein in human pancreatic β cells, ShK has no effect on the Kv current of these cells, suggesting that it is unlikely that homotetrameric Kv3.2 channels contribute significantly to the delayed rectifier current of insulin-secreting cells. In mouse cortical GABAergic fast-spiking interneurons, however, application of ShK produced effects consistent with the blockade of Kv3 channels (i.e., an increase in action potential half-width, a decrease in the amplitude of the action potential after hyperpolarization, and a decrease in maximal firing frequency in response to depolarizing current injections). Taken together, these results indicate that ShK is a potent inhibitor of Kv3.2 channels and may serve as a useful pharmacological probe for studying these channels in native preparations.

Voltage-gated potassium channels (Kv) represent a large family of proteins that regulate numerous physiological functions. Kv channels are tetrameric structures formed by the association of identical or closely related subunits. The study of potassium channels has been greatly facilitated by the discovery of high-affinity and selective channel inhibitors in the venoms of different organisms (Garcia et al., 1998; Corzo and Escoubas, 2003; Lewis and Garcia, 2003). Such peptidyl inhibitors have provided invaluable pharmacological tools for the isolation, purification, tissue localization, and the study of the structure and gating mechanism of ion channels (Garcia-Calvo et al., 1994).

To date, many subtype-selective Kv channel inhibitors have been identified. Dendrotoxins (DTXs) from the venom of Dendroaspis angusticeps and BgK from the sea anemone Bunodosoma granuliferaare are potent blockers of Kv1.1, 1.2, and 1.6 channels (Harvey, 2001). Charybdotoxin (ChTX), a minor component of Leuiurus quinquestriatus venom, is a potent blocker of Kv1.3 and Kv1.2 channels (Castaneda et al., 1995; Garcia et al., 1999), whereas ShK, a peptide isolated from the sea anemone Stichodactyla helianthus, inhibits Kv1.1 and 1.3 channels with picomolar affinities (Middleton et al., 2003). Blood-depressing substances I and II from the venom of sea anemone Anemonia sulcata block Kv3.4 channels specifically (Diochot et al., 1998). Finally, phrixotoxins I and II from venom of the tarantula Phriotrichus auratus and hanatoxin from the venom of the spider Grammostola spatulata represent a class of gating-modifying peptides with activity against Kv4.2 and 4.3 (Diochot et al., 1999) and Kv2.1 channels (Swartz and MacKinnon, 1995), respectively.

Kv3.2 is a member of the Kv3 channel subfamily. These channels possess rather unique fast activation at voltages positive to –20 mV and very fast deactivation rates (Coetzee et al., 1999). Such biophysical properties could enable cells to fire high-frequency trains of action potentials, and therefore Kv3.2 could play a role in neuronal excitability. Consistent with this proposal, Kv3.2 is expressed in neurons that are known to fire at a high frequency, such as the inhibitory cortical GABAergic interneurons (Chow et al., 1999; Hernandez-Pineda et al., 1999; Tansey et al., 2002). In addition, Kv3.2 is expressed in the insulin-secreting, pancreatic β cells (Yan et al., 2004) and could contribute to the delayed-rectifier current in these cells. The β cell delayed-rectifier current is believed to regulate glucose-dependent firing, making it an attractive candidate for the development of novel glucose-dependent insulin secretagogues that could have usefulness in the treatment of type-2 diabetes. Although Kv3.2 channels are sensitive to low concentrations of tetraethylammonium ion (TEA) (Hernandez-Pineda et al., 1999), potent and selective Kv3.2 inhibitors with which to study the role of these channels in native tissues are not currently available.

To search for Kv3.2 inhibitors, we generated CHO-K1 cells stably expressing the human Kv3.2b channel (CHO-K1.hKv3.2b cells) and established a functional 86Rb+ efflux assay with which to monitor the activity of the channel. Using this assay, ShK was identified as a potent inhibitor (IC50 = 0.59 nM) of heterologously expressed hKv3.2b channels and was used to evaluate the role of the channel in pancreatic β cells and in cortical GABAergic fast-spiking interneurons. ShK may serve as a useful pharmacological tool for studying Kv3.2 channels in native tissues.

Conventional Patch-Clamp Electrophysiology. Membrane currents were recorded using standard whole-cell voltage-clamp techniques (Hamill et al., 1981) with a Dagan 3900A amplifier (Dagan, Minneapolis, MN). Microelectrodes fabricated from borosilicate glass were coated with Sylgard (Dow Corning, Midland, MI) and fire-polished. Electrode resistances were generally 2 to 4 MΩ when filled with standard internal saline. The reference electrode was a silver-silver chloride wire within an agar bridge (4% agar in 170 mM KCl). Voltages given in the figures have not been corrected for the liquid junction potential between the internal and external solutions. All experiments were performed at room temperature (22–25°C). Generation of analog voltage commands and digitization of membrane currents were controlled with PULSE software (HEKA Elektronik, Lambrecht, Germany) and an ITC-16 computer interface (InstruTECH Corporation, Port Washington, NY). Currents were digitized at 5 kHz and digitally filtered at 2 kHz. Digital subtraction of leakage and capacitive currents was performed by the P/n procedure, where n = 5. Voltage steps were applied every 5 to 30 s from a holding potential of –80 mV.

86Rb+ Efflux Assay. CHO-K1 hKv3.2b cells were plated into 96-well cell culture plates at a density of ∼2 × 105 cells/well and incubated with 6 μCi/ml 86RbCl in 100 μl of culture medium overnight at 37°C. To each well, 100 μl of low-potassium buffer (135 mM NaCl, 4.6 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 0.2% bovine serum albumin, and 10 mM HEPES, pH 7.4, with NaOH) with or without test samples was added, and incubation continued for 30 min at 37°C. At the end of the incubation, the medium was replaced with 200 μl of high-potassium buffer (140 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 0.2% bovine serum albumin, and 10 mM HEPES, pH 7.4, with KOH), with or without test samples, and 86Rb+ efflux was monitored for determined periods of time. To quantify the amount of 86Rb+ efflux, medium was removed from the wells, and 100 μl was mixed with 100 μl of MicroScint-20 (PerkinElmer Life and Analytical Sciences). 86Rb+ content of the cells was determined by the addition of 100 μl of MicroScint-20. Efflux was defined as the percentage of 86Rb+ in each well in the low- or high-potassium buffer, normalized to the total radioactivity of the efflux solution and cells. IC50 values represent the mean ± S.E.M.

Recordings were made at room temperature in modified ND-96 consisting of 96 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 3.5 mM BaCl2, and 5 mM HEPES, pH 7.5. Oocytes were voltage-clamped using a Dagan CA1 two-microelectrode amplifier (Dagan Corporation) interfaced to a Macintosh 7100/80 computer (Apple Computer, Cupertino, CA). The current-passing electrode was filled with 0.7 M KCl and 1.7 M potassium citrate, and the voltage-recording electrode was filled with 1 M KCl. Throughout the experiment, oocytes were superfused with modified ND-96 (control solution) or with ND-96 containing test peptides at a rate of approximately 3 ml/min. Data were acquired at 5 kHz and filtered at 1.7 kHz using Pulse software (version 8.53) from HEKA Elektronik. All recordings were performed from a holding potential of –80 mV. IC50 values represent the mean ± S.D.

Planar Patch-Clamp Electrophysiology. Parallel patch-clamp electrophysiology was performed essentially as described previously (Kiss et al., 2003). In brief, CHO-K1.hKv3.2b cells or CHO-K1.hKv2.1 cells were removed from culture plates using Versene (Invitrogen), subjected to centrifugation, and resuspended in 4 ml of Dulbecco's phosphate-buffered saline (Invitrogen) containing 138 mM NaCl, 8.1 mM Na3PO4, 2.67 mM KCl, 1.47 mM K3PO4, 0.9 mM CaCl2, and 0.5 mM MgCl2, pH 7.4. The internal solution consisted of 100 mM potassium gluconate, 40 mM KCl, 3.2 mM MgCl2, 3 mM EGTA, and 5 mM HEPES, pH 7.4, with KOH, supplemented with 120 μg/ml amphotericin B. Voltage protocols and the recording of membrane currents were performed using the IonWorks HT software/hardware system (Molecular Devices, Sunnyvale, CA). Currents were sampled at 2.5 kHz, and leakage subtraction was performed using a 10-mV step from the holding potential (–80 mV) and assuming a linear leak conductance. No correction for liquid junction potentials was used. Wells with seal resistances less than 70 MΩ or less than 0.1 nA of Kv current at the test potential (+40 mV) were excluded from analysis. The mean seal resistance of the remaining wells was 169 MΩ. Current amplitudes were calculated with the IonWorks software, fraction block was calculated in Microsoft Excel (Microsoft, Redmond, CA), and fitting was performed with Sigma Plot (SPSS Inc., Chicago, IL).

Human Islet Cells. Human pancreatic islets were obtained from Dr. Jonathan Lakey (University of Alberta, Surgical-Medical Research Institute, Edmonton, AB, Canada) and the JDRF Human Islet Distribution Program at the University of Alberta (Edmonton, AB, Canada). Islets were isolated as described previously (Shapiro et al., 2000) using procedures approved by the University of Alberta Ethics Committee. Upon receipt of the tissue, islets were hand-picked, dissociated into single cells using trypsin, resuspended in culture medium, and plated on poly-l-lysine–coated glass chips. Cells were used for electrophysiology within 72 h of plating.

Results

Stable Expression of hKv3.2b in CHO-K1 Cells. To establish a robust, functional assay for identifying hKv3.2 inhibitors, we stably expressed the splice variant, hKv3.2b, in CHO-K1 cells. CHO-K1 cells were chosen because it is known that these cells express low levels of endogenous potassium channels, and they are routinely used for functional heterologous expression of Kv channels. Figure 1A shows a Western blot analysis of the CHO-K1.hKv3.2b cell clone that was selected for all subsequent studies. Two bands with estimated molecular masses of ∼57.5 and 74 kDa are specifically recognized by the Kv3.2 antibody in the lysate of these cells (lane 2) but not in the lysate of the parental, untransfected CHO-K1 cells (lane 1). The upper band agrees well with the predicted molecular mass of the full-length protein, whereas the lower band probably represents a proteolytic product of the higher molecular mass product.

Stable expression of hKv3.2b protein in CHO-K1 cells. A, Western blot analysis of protein extracts from the parental, untransfected CHO-K1 cells (lane 1) or from CHO-K1.hKv3.2b cells (lane 2). Equal amounts of total protein (∼50 μg) were loaded onto each lane, and blots were probed with a rabbit polyclonal antibody raised against a sequence of Kv3.2b. The position of the molecular mass markers is indicated. B to F, whole-cell patch-clamp recordings from CHO-K1.hKv3.2b cells. B, outward currents in response to 100-ms step depolarizations ranging from –60 to +60 mV (in 10-mV increments) from a holding potential of –80 mV. C, voltage dependence of activation for the same cell shown in B determined from the amplitude of tail currents. The solid line is a fit of the Boltzmann equation to the data. Parameters of the fit are given in the text. D, inactivation of hKv3.2b currents by long depolarizations (7 s) ranging from –20 to +60 mV (in 10-mV increments). E, plot of the peak current amplitude in response to 100-ms step depolarizations to +20 mV from a holding potential of –80 mV versus time. The periods of application of 0.3, 1, or 0.1 mM TEA are denoted by the solid bars. F, dose-response relationship for TEA on hKv3.2b current. Average fraction block (± S.E.M.) is plotted versus TEA concentration (n = 4 per concentration). The solid line is a fit of the Hill equation to the data with an apparent IC50 value of 0.3 mM and a slope of 1.0.

Electrophysiological Properties of CHO-K1.hKv3.2b Cells. The biophysical properties of heterologously expressed hKv3.2b channels were evaluated in patch-clamp recordings. Characteristics of the Kv3 channel subfamily (including Kv3.2) include activation at potentials more positive than approximately –20 mV and fast deactivation upon repolarization (Rudy and McBain, 2001). Whereas the parental untransfected CHO-K1 cells display little if any Kv current (data not shown), CHO-K1.hKv3.2b cells displayed large outward currents in response to step depolarizations (Fig. 1B). The currents activated rapidly (τ = 3.0 ± 0.9 ms at +40 mV, n = 5) and deactivated rapidly (τ = 5.6 ± 0.2 ms at –40 mV, n = 5). The voltage dependence of activation was studied by measuring the amplitude of tail currents after step depolarizations to various potentials. For the cell shown in Fig. 1B, the voltage-dependence of activation was well-described by a Boltzmann distribution with a V50 of +22.6 mV and a slope of 13.5 mV (Fig. 1C). On average, the V50 was +20.2 ± 0.9 mV and the slope was 14.2 ± 0.5 mV (n = 5). To study current inactivation, long depolarizations (7 s) to various potentials were given (Fig. 1D). Currents decayed slowly at all potentials tested. At +40 mV, the current decayed 28 ± 7% (n = 3) over the 7-s step. Kv3 currents are known to be sensitive to low concentrations of TEA (Hernandez-Pineda et al., 1999), and this was also true for the channels present in CHO-K1.hKv3.2b cells (Fig. 1E). In these experiments, TEA reversibly inhibited hKv3.2b currents in a dose-dependent fashion. The combined data from experiments carried out in four cells illustrate that TEA blocked hKv3.2b with an IC50 of 0.3 mM (Fig. 1F). Taken together, the data indicate that CHO-K1.hKv3.2b cells display a TEA-sensitive current with characteristics consistent with Kv3.2.

TEA-Sensitive 86Rb+ Efflux from CHO-K1.hKv3.2b Cells. A functional, high-capacity assay for identifying hKv3.2 inhibitors was developed on the basis of the ability of 86Rb+ to permeate through potassium channels. Cells incubated with 86RbCl accumulate 86Rb+ through operation of the Na/K-ATPase pump. When CHO-K1.hKv3.2b cells were then exposed to physiological, low external potassium (low-K) conditions, a time-dependent efflux of 86Rb+ from the cells occurred. In the presence of high external potassium, however, 86Rb+ efflux occurred much more rapidly, reflecting the activity of Kv channels. The Kv channel-mediated component of 86Rb+ efflux, defined as the difference between efflux under high-K and low-K conditions, reached a plateau in approximately 10 min (Fig. 2A). 86Rb+ efflux from CHO-K1.hKv3.2b cells was inhibited by TEA in a dose-dependent manner with an IC50 of 0.25 ± 0.01 mM (n = 3) (Fig. 2B). High-K dependent 86Rb+ efflux was not observed in the parental, untransfected CHO-K1 cells, although the basal levels of efflux in low-K were similar to those of CHO-K1.hKv3.2b cells (data not shown). These data suggest that in CHO-K1.hKv3.2b cells, high-K–induced 86Rb+ efflux reflects the activity of hKv3.2 channels. The high-capacity, 86Rb+ functional assay provides a means for identifying Kv3.2 inhibitors.

86Rb+ efflux from CHO-K1.hKv3.2b cells. A, time course of 86Rb+ efflux from CHO-K1.hKv3.2b cells in low-K (•) and high-K (○) conditions. The Kv3.2 component of 86Rb+ efflux, defined as the difference between high- and low-K, is indicated (▾). B, inhibition of 86Rb+ efflux from CHO-K1.hKv3.2b cells by TEA. The Kv3.2 component of 86Rb+ efflux at 10 min is plotted relative to the concentration of TEA. Each point represents the average of four replicates. The solid line is a fit of the Hill equation with an apparent IC50 value of 0.24 nM. C, concentration-dependent inhibition of 86Rb+ efflux from CHO-K1.hKv3.2b cells by ShK (○) and ShK-Dap22 (•). The Kv3.2 component of 86Rb+ efflux at 10 min is plotted against the concentration of peptide. Each data point is the average of three replicates. The solid lines are fits of the Hill equation with apparent IC50 values of 0.59 (○) and 57 (•) nM, respectively.

ShK Is a Potent Inhibitor of hKv3.2b Channels. Using the 86Rb+ efflux assay described above, we evaluated a number of scorpion venoms and several known potassium channel inhibitors for their ability to block hKv3.2b channels. All venoms and the majority of potassium channel inhibitors such as apamin, kaliotoxin, iberiotoxin, agitoxin-I, agitoxin-II, margatoxin, and ChTX, when tested at concentrations up to 300 nM, did not significantly inhibit 86Rb+ efflux from the CHO-K1.hKv3.2b cells (data not shown). In marked contrast, 100 nM ShK caused complete inhibition of 86Rb+ efflux, and ShK-Dap22, in which diaminopropionic acid (Dap) was substituted at lysine 22 of ShK (Kalman et al., 1998), also inhibited 86Rb+ efflux to a lesser extent. Dose-response curves for inhibition of 86Rb+ efflux from CHO-K1.hKv3.2b cells indicate that ShK and ShK-Dap-22 block hKv3.2b-mediated 86Rb+ efflux with IC50 values of 0.62 ± 0.10 (n = 3) and 83.2 ± 22.1 (n = 3) nM, respectively, and Hill coefficients of 1 (Fig. 2C). These results provide the first evidence that ShK is a potent inhibitor of hKv3.2b channels.

ShK Inhibits hKv3.2b but Not hKv2.1 Channels. Because both Kv2.1 and Kv3.2 channels are expressed in human pancreatic β cells and either one or both channels could contribute to the delayed-rectifier potassium current that regulates the firing frequency of the cells in the presence of high glucose, we compared the ability of ShK to inhibit both channels. X. laevis oocytes expressing hKv3.2b exhibited rapidly activating currents in response to step depolarizations to +20 and +80 mV (Fig. 3A) that were not seen in uninjected or water-injected oocytes. Similar to hKv3.2b currents expressed in mammalian cell lines, the hKv3.2b current in oocytes was blocked by TEA with an IC50 of 0.10 ± 0.01 mM (n = 3) (data not shown). Bath application of 0.3 nM ShK reduced the current activated by depolarizations to +20 and +80 mV (Fig. 3A, gray line). This inhibition of current amplitude was concentration-dependent, shown in Fig. 3B for the current at +80 mV, and displayed an IC50 value of 0.31 ± 0.06 nM (n = 6).

ShK inhibits hKv3.2b but not hKv2.1 channels. A, hKv3.2b currents expressed in X. laevis oocytes in response to depolarizations to +20 and +80 mV in control (black trace) and in 0.3 nM ShK (gray trace). B, time course of block of hKv3.2b currents, activated at +80 mV, by ShK. C, concentration-dependent inhibition of hKv3.2b currents by ShK for the cell shown in B. The solid line is a fit of the Hill equation with an apparent IC50 value of 0.5 nM and a slope of 1. D, dose-response relationship for the inhibition of hKv3.2b (○) and Kv2.1 (▪) by ShK studied with a planar patch-clamp device (IonWorks HT). The solid line is a fit of the Hill equation to the hKv3.2b data with an apparent IC50 value of 6 nM and a slope of 1. In contrast, hKv2.1 was not blocked by 1 μM ShK.

In an automated patch-clamp system (IonWorks HT) that performs synchronous perforated patch recordings in a 384-wells planar array (Schroeder et al., 2003), the blocking potency of ShK against hKv3.2b or Kv2.1 channels was evaluated after a 10-min incubation period in either peptide or vehicle. ShK inhibited hKv3.2b current in a dose-dependent manner with an IC50 value of 6 nM but had no effect on hKv2.1 current at concentrations up to 1 μM (Fig. 3D). These results suggest that ShK can be used as a pharmacological tool to evaluate the contribution of Kv3.2 channels to the whole-cell currents of cells that express both Kv3.2 and Kv2.1 channels. One such cell type is the human pancreatic β cell.

ShK does not inhibit the Kv current of human pancreatic beta cells. A, plot of the peak current amplitude in response to 100-ms step depolarization to +20 mV from a holding potential of –80 mV versus time. The period of application of 100 nM ShK is denoted by the solid bar. B, representative current traces before and during application of ShK. C and D, effect of 1 and 10 mM TEA on peak Kv current for a different cell.

The Effects of ShK on Fast-Spiking Cortical Interneurons Are Consistent with Block of Kv3.2 Channels. The presence of Kv3 channels in cortical GABAergic FS cells is required for the rapid repolarization of action potentials as well as for the initiation and maintenance of high-frequency firing. Application of low concentrations of TEA (a nonselective blocker of Kv3 channels) or genetic elimination of Kv3 channels produces broadening of the FS cell action potential, ablation of the afterhyperpolarization (AHP), and an attenuation of maximal achievable firing frequencies by these cells (Erisir et al., 1999; Lau et al., 2000; Rudy and McBain, 2001). Bath application of 100 nM ShK had no effect on resting membrane potential (Vm; –73.0 ± 5.5 and –75.0 ± 5.2 mV in control and after addition of ShK, respectively; n = 4) but produced a small increase in membrane resistance (Rm) from 112 ± 52 MΩ under control conditions to 139 ± 61 MΩ after ShK application (an increase of 26 ± 20% over control; n = 4). However, consistent with blockade of Kv3 channels, bath application of 100 nM ShK produced an increase in action potential half-width (i.e., the width of the action potential at half-amplitude, measured from action potential threshold to action potential peak) from 0.41 ± 0.05 to 0.58 ± 0.16 ms (n = 4), a decrease in the amplitude of the AHP as measured from action potential threshold to the maximal negativity of the afterhyperpolarization from 19.9 ± 8.1 to 14.1 ± 8.0 mV (n = 4), and a decrease in maximum firing frequency in response to depolarizing current injections from 280 ± 53 to 181 ± 93 Hz (n = 4) (Fig. 5). Statistical significance at the p < 0.05 level was achieved via unpaired, two-tailed t test for AHP and maximal firing frequency but not for the other parameters shown in Fig. 5F. In contrast, 100 nM dendrotoxin-I, a selective inhibitor of Kv channels containing Kv1.1, Kv1.2, and/or Kv1.6 subunits, has no effect on the FS cell action potential and, in fact, produces a slight increase in firing frequency (Erisir et al., 1999). However, ShK did produce a marked increase in the frequency of spontaneous postsynaptic potentials recorded from FS cells (data not shown). This effect is qualitatively similar to that produced by 100 nM dendrotoxin-I and is consistent with blockade of Kv1 channels, probably present in the presynaptic terminals of intracortical excitatory synapses (Coetzee et al., 1999; Bekkers and Delaney, 2001; Harvey, 2001).

The effects of ShK on neocortical FS cells are consistent with blockade of Kv3 channels. A, firing pattern of an FS cell in response to 300-ms hyper- and depolarizing current injections under control conditions. Note that the cell discharges with a sustained, nonadapting train of high-frequency action potentials. B, same cell after bath application of 100 nM ShK. C, the first action potential of the first suprathreshold sweep for control (black) and after bath application of 100 nM ShK (red). Note that ShK produces spike-broadening and attenuates the fast, deep afterhyperpolarization characteristic of the FS cell action potential. D, for all cells tested, bath application of 100 nM ShK produced an increase in the AP half-width (AP ½-width). E, same as D except for amplitude of the AHP (as measured from action potential threshold). F, summary data. Consistent with blockade of Kv3 channels, bath application of 100 nM ShK produced an increase in AP ½-width (from 0.41 ± 0.05 to 0.58 ± 0.16 ms; p = 0.066, n = 4), a decrease in the amplitude of the AHP (from 19.9 ± 8.1 to 14.1 ± 8.0 mV; p = 0.028, n = 4) and a decrease in maximum firing frequency (from 280 ± 53 to 181 ± 93 Hz; p = 0.040, n = 4).

Discussion

The goal of the present study was to identify potent and selective inhibitors of Kv3.2 channels with which to investigate the physiological role of the channel in native preparations. To this end, we constructed a CHO-K1.hKv3.2b cell line that stably expresses hKv3.2b channels and established a functional 86Rb+ efflux assay that was used to screen a variety of venoms and known potassium-channel inhibitors. ShK, a peptide isolated from S. helianthus venom, was found to inhibit hKv3.2b-mediated 86Rb+ efflux from CHO-K1.hKv3.2b cells in a dose-dependent manner and with high potency (IC50 = 0.62 nM). In electrophysiological recordings from X. laevis oocytes expressing hKv3.2b channels, ShK displayed an IC50 of 0.31 nM for channel inhibition, whereas in CHO-K1.hKv3.2b cells, the IC50 was ∼6 nM measured by planar patch-clamp electrophysiology. Taken together, these results suggest that ShK is a potent inhibitor of heterologously expressed hKv3.2b channels.

ShK is a potent inhibitor of certain members of the Kv1-channel family. In particular, ShK blocks Kv1.1 and Kv1.3 channels with IC50 values of ∼1 pM, a 1000-fold lower concentration than that required to inhibit Kv3.2 channels. Evidence also suggests that native Kv currents in the central nervous system, which are predominantly carried by Kv1.2 channels, are highly sensitive to the peptide (Middleton et al., 2003). Thus, the usefulness of ShK in discerning the physiological role of Kv3.2 channels must be considered with caution in preparations in which other ShK-sensitive channels may be present. This is particularly relevant to the central nervous system, given the large diversity of Kv channels that exist in different neurons and the wide distribution of Kv1 subunits. Nonetheless, in cells in which Kv1 channels are present, other Kv1 specific peptides, such as DTX, could be used to distinguish between Kv3- and Kv1-mediated effects of ShK. In other systems, in which channel distribution is more restricted and better defined, ShK provides a pharmacological tool for studying Kv3.2 channels. For instance, polymerase chain reaction, in situ hybridization, and immunostaining with specific Kv antibodies have provided strong evidence for the presence of Kv2.1- and Kv3.2-channel subunits in pancreatic β cells (MacDonald et al., 2002; Yan et al., 2004). Because the association of Kv2.1 and Kv3.2 subunits has not been demonstrated either in vitro or in vivo, the delayed-rectifier Kv current in β cells is likely to be carried by channels that contain either Kv2.1 or Kv3.2 subunits. Defining the subunit composition of the delayed-rectifier current in β cells is important because this current regulates firing frequencies in the presence of glucose, and inhibitors of this current would be expected to enhance glucose-dependent insulin secretion and therefore have usefulness in the treatment of type-2 diabetes. ShK does not inhibit hKv2.1 currents, and because Kv1.1 subunits are not found in human pancreatic islets and functional Kv1.3 channels have not been reported in human β cells (MacDonald and Wheeler, 2003; Yan et al., 2004), the putative contribution of Kv3.2 subunits to the β-cell Kv current can be evaluated. Even when high concentrations of ShK were used, no inhibition of the human delayed-rectifier current in β cells was observed. Although these experiments strongly suggest that a homomultimeric Kv3.2 channel is not a component of the β cell delayed-rectifier current, it raises the question of why the channel subunit is expressed in these cells. It is possible that coexpression of Kv3.2 with other ShK-insensitive subunits could lead to a tetrameric complex insensitive to the peptide, and additional experiments are needed to evaluate this possibility.

Cortical GABAergic FS cells are named for their ability to fire sustained trains of brief action potentials at high frequency and, in fact, display the highest firing rates of any cortical cell (McCormick et al., 1985; Connors and Gutnick, 1990). Cortical FS cells express Kv3.1 and Kv3.2 subunits. A large body of evidence now exists to support the conclusion that the fast-spiking phenotype is, in fact, determined by the somatic expression of these Kv3 channels (Rudy and McBain, 2001; Lien and Jonas, 2003), which activate at depolarized potentials (relative to other Kv channels) and deactivate rapidly. Even at high firing frequencies, the effect of activation of Kv3.1/Kv3.2 channels is largely restricted to the repolarization phase of the action potential (Du et al., 1996), with little direct effect on the rate of rise or on spike threshold, and the interspike interval is established to a large degree by the accumulation of voltage-gated sodium-channel inactivation. However, by increasing the rate of spike repolarization (and keeping action potentials brief), Kv3.1/Kv3.2 channels limit sodium-channel inactivation. Moreover, the large, fast afterhyperpolarization elicited by Kv3.1/Kv3.2 currents accelerates recovery from inactivation of sodium channels (Rudy and McBain, 2001). Thus, Kv3.1/Kv3.2 channels ensure that sufficient sodium channels are available for a subsequent action potential, with little counteractive Kv current. The effects of pharmacological blockade or genetic elimination of Kv3.1/Kv3.2 channels has provided strong evidence that these channels are critical for high-frequency repetitive firing in FS cells (Martina et al., 1998; Erisir et al., 1999; Lau et al., 2000), as has a recent study using dynamic clamp technology (Lien and Jonas, 2003). In this context, the results of this study concerning the effect of ShK in FS cells are consistent with the proposed role for Kv3.2 channels and support the use of the peptide for studying these channels in other native preparations. Contribution of Kv1.1 and/or Kv1.3 channels to the observed effects of ShK in FS cells does not seem likely because DTX-I and ChTX, potent inhibitors of Kv1.1 and Kv1.3 channels, respectively, do not reproduce the effects of ShK on the shape of the FS cell action potential and on repetitive firing. In addition, Kv1.3 channels with their very slow recovery from inactivation would not be able to contribute to the fast firing frequency of FS cells.

A significant difference in the inhibitory potency of ShK on Kv3.2b channels was observed between X. laevis oocyte electrophysiology (IC50 = 0.31 nM) and planar patch-clamp electrophysiology on CHO-K1.hKv3.2b cells (IC50 = 6 nM) (Fig. 3, C and D). Indeed, IC50 values for inhibition of 86Rb+ flux from CHO-K1.hKv3.2b cells are closer to those obtained from electrophysiological experiments on oocytes. Although the reasons for the block affinity of ShK in the planar patch-clamp system are not fully understood, several possibilities, such as an incomplete equilibration and adsorption to the planar array caused by the high surface-to-volume ratio, could account for the observed differences.

In summary, we have provided evidence that ShK is a potent inhibitor of the hKv3.2b channel. Although much remains to be learned about the structural basis of the interaction between ShK and the Kv3.2 channel, it is likely that ShK will serve as an important pharmacological tool for the study of Kv3 family channels in native systems, particularly in those situations in which Kv1 channels are not present or can be selectively modulated.

Diochot S, Drici MD, Moinier D, Fink M, and Lazdunski M (1999) Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis. Br J Pharmacol126:251–263.